HIV enters cells through sequential interactions of the viral envelope (Env) glycoprotein with the cell surface protein CD4 and a chemokine receptor CXCR4 or CCR5. The viral envelope gp120-gp41 heterodimers associate in a trimer to form spikes on the viral surface. Structural studies suggest that a mature HIV particle contains 72 spikes and the distance between two spikes is 21-22 nm, and a spike forms a knob with a diameter of 14 nm. Binding of CD4 to gp120 leads to conformational changes in a spike that allows the gp120 to interact with a chemokine receptor. Ligation of gp120 to CD4 and a chemokine receptor triggers further structural changes that allow the gp41 to insert into the target cell membrane. In the current model, one gp120 interacts with one CD4 and one chemokine receptor. Evidence from immunoelectron microscopy demonstrated that before contacting HIV, CD4, CXCR4 and CCR5 each form independent microclusters that are separated by a distance of about 10 nm. Based on structural and immuno-EM studies, we estimate that the distance between CD4 and the chemokine receptors is well within 10nm when a HIV entry complex is formed. Our favored hypothesis is that when gp120 trimers bind CD4 molecules, gp120-CD4 complexes induce the association of chemokine receptors with the trimers to form productive HIV entry complexes. Colocalization of CD4 and CXCR4 or CCR5 induced by gp120 has been shown using immuno-staining. However, due to resolution limitations of immuno-staining, the temporal and spatial arrangement and physical interactions between CD4 and the chemokine receptors are still unclear. [unreadable] [unreadable] We have employed fluorescence resonance energy transfer (FRET) between chimeras of CD4, CXCR4 and CCR5 fused with CFP or YFP to probe their membrane distribution and to visualize molecular interactions of CD4 and the chemokine receptors in living cells. We have also used fluorescence recovery after photobleaching (FRAP) to measure lateral diffusion of CD4 and CCR5 that each fussed with CFP or YFP in living cell membrane to probe their spatial distribution. We found that gp120 induced FRET increase between CD4-YFP and CCR5-CFP, suggesting that HIV envelope protein promotes association between CD4 and CCR5 on the cell membrane. The plasma membranes consist of a complex assembly of various lipids and proteins that are distributed in regions of distinct lipid microenvironments, known as lipid raft or non-raft microdomains. Lipid rafts are defined as microdomains that are enriched in cholesterol, glycosphingolipid and sphingomyelin. Both lipid and non-lipid raft microdomains contain multiple proteins that play critical roles in signal transduction via complex protein-protein interactions between ligands, receptors, and signaling components. To examine whether lipid raft microenvironment is essential for gp120-induced association between CD4 and CCR5, we disrupted these microenvironments on the plasma membrane by depleting cholesterol with MRCD and measured FRET between CD4-YFP and CCR5-CFP. We found interestingly that depleting cholesterol had no effect on gp120-promoted association between CD4 and CCR5. Using FRAP measurement, we showed that CD4 is more mobile than CCR5 on the plasma membrane. However, when both CD4 and CCR5 were expressed on the membrane, their mobility became similar, suggesting some interactions between these two receptors on the membrane even in the absence of gp120. We are in the process to complete FRET and FRAP experiments, which may provide insight into molecular interactions between CD4 and the chemokine receptors during HIV entry.